The regulatory mechanism of CCR7 gene expression and its involvement in the metastasis and progression of gastric cancer.

Gastric cancer is the second leading cause of cancer mortality, but the molecular mechanisms underlying its progression and metastasis remain unclear. CCR7 and Dicer 1 protein expression in 80 gastric adenocarcinomas and 40 peritumoral tissues were measured by immunohistochemical staining. The expression of let-7a miRNA in serum, tumor tissues, and peritumoral tissues was measured by real-time PCR. The role of let-7a in CCR7 protein expression, migration, and invasion of gastric cancer cells was tested in vitro. Dicer 1 protein expression was found to be significantly reduced, whereas CCR7 protein expression was significantly increased in gastric adenocarcinomas compared to peritumoral tissues. The let-7a miRNA levels in the serum and tumor tissues of gastric adenocarcinoma patients were significantly lower than in the serum of healthy controls and peritumoral tissues, respectively. Dicer 1 protein positively correlated with let-7a miRNA level, but negatively correlated with CCR7 protein level in gastric adenocarcinoma. Negative Dicer 1 protein and let-7a miRNA expression and positive CCR7 protein expression significantly correlated with lymph node metastasis, depth of invasion, high clinical TNM stage, and larger tumor size. Let-7a transfection significantly inhibited CCR7 protein expression, migration, and invasion of MNK-45 cells in vitro. High expression of CCR7 protein and low expression of Dicer 1 protein and let-7a miRNA are significantly associated with the metastasis and progression of gastric cancer. High CCR7 protein expression may be caused by the loss of Dicer 1 protein expression and reduced let-7a miRNA level in gastric cancer. The serum let-7a level might be a marker for the diagnosis of gastric cancer.

Calcification resistance for photooxidatively crosslinked acellular bovine jugular vein conduits in right-side heart implantation.

This study aimed to investigate the effect of decellularization plus photooxidative crosslinking and ethanol pretreatment on bioprosthetic tissue calcification. Photooxidatively crosslinked acellular (PCA) bovine jugular vein conduits (BJVCs) and their photooxidized controls (n = 5 each) were sterilized in a graded concentration of ethanol solutions for 4 h, and used to reconstruct dog right ventricular outflow tracts. At 1-year implantation, echocardiography showed similar hemodynamic performance, but obvious calcification for the photooxidized BJVC walls. Further histological examination showed intense calcium deposition colocalized with slightly degraded elastic fibers in the photooxidized BJVC walls, with sparsely distributed punctate calcification in the valves and other areas of walls. But PCA BJVCs had apparent degradation of elastic fibers in the walls, with only sparsely distributed punctate calcification in the walls and valves. Content assay demonstrated comparable calcium content for the two groups at preimplantation, whereas less calcium for the PCA group in the walls and similar calcium in the valvular leaflets compared with the photooxidized group at 1-year retrieval. Elastin content assay presented the conduit walls of PCA group had less elastin content at preimplantation, but similar content at 1-year retrieval compared with the photooxidized group. Phospholipid analysis showed phospholipid extraction by ethanol for the PCA group was more efficacious than the photooxidized group. These results indicate that PCA BJVCs resist calcification in right-side heart implantation owing to decellularization, further photooxidative crosslinking, and subsequent phospholipid extraction by ethanol at preimplantation.

The performance of photooxidatively crosslinked acellular bovine jugular vein conduits in the reconstruction of connections between pulmonary arteries and right ventricles.

In this study, valved photooxidatively crosslinked acellular bovine jugular vein conduits (BJVCs) were implanted in young dogs to reconstruct the connections of pulmonary arteries and right ventricles, with acellular conduits used as controls. All acellular conduits had moderate to severe valvular dysfunction and were explanted at 1-month implantation (n = 5). Histological examination showed inflammatory cell infiltration and intimal hyperplasia in the walls, and severe inflammatory cell infiltration and thrombosis in the valves. The photooxidatively crosslinked acellular conduits were retrieved at 1-month (n = 5) and 6-month (n = 5) implantations respectively. These conduits had excellent valvular function at retrieval. Their walls and valves were still soft and smooth without calcification and hemangioma. Endothelialization in valves and luminal walls was unsatisfied at 1-month retrieval, and was improved at 6-month retrieval. Host cells infiltrated and migrated from outer layer to the middle layer, with tissue remolding and regeneration found in these recellular regions. Histological examination and tissue content assay demonstrated that degeneration and regeneration of collagens and glycosaminoglycans were comparable, but elastic fibers gradually degraded. Photooxidatively crosslinked acellular BJVCs resist calcification and thrombosis and have regeneration patterns, with excellent hemodynamic performance.

Decellularized and photooxidatively crosslinked bovine jugular veins as potential tissue engineering scaffolds.

Decellularization means and altering crosslinking approaches were two promising alternatives for glutaraldehyde fixation to biological tissues. Bovine jugular veins (BJVs) were decellularized by a multi-step detergent-enzymatic extraction method, then photooxidatively crosslinked. Gross and histological integrity of which was retained. Ultrastructures showed integrity of collagen fibrils and elastic fibers, and a basement membrane free luminal surface. Mechanical strength test and tissue protein extraction assay demonstrated their tissue stability. After being pre-coated with gelatin, collagen IV and fibronectin, cultured human umbilical vein endothelial cells were planted in the luminal surface of decellularized plus photooxidized BJV patches for seven days. Endothelial cells were denser in pre-coated patches than in uncoated controls. A rat subcutaneous implantation model revealed more resistance against in vivo degradation for further crosslinked BJV patches than decellularized patches at 12-week retrieval. Host cells were all layer repopulated for both. Histological examination and content assay demonstrated collagen and glycosaminoglycan components synthesis for decellularized plus photooxidized BJV patches. Decellularized and photooxidatively crosslinked BJV patches possess tissue integrity, excellent in vitro and in vivo tissue stability and repopulation patterns. Thus, they have potentials as tissue engineering scaffolds in future cardiovascular surgery.

[Effect of decellular treatment on the framework of extracellular matrix in bovine jugular vein conduit].

OBJECTIVE: To investigate the effect of decellular treatment on the framework constituents of extracellular matrix and tissue stability in bovine jugular vein conduit (BJVC), and to provide an evidence for tissue engineering of vascular prosthesis. METHODS: Bovine jugular veins were obtained fresh from a local slaughterhouse and were stored in chilled PBS. In the laboratory, any fat and loose connective tissue on the outer surface of the vessel was trimmed. BJVCs were decellularized by a 3-step extraction method as detergent Triton X-100 (0.5%), Trypsin (0.025%) EDTA (0.02%), and DNase I(30kU/L) RNaseA(0.3g/L). Histological and transmission electron microscopy (TEM) techniques were used to study the framework constituents of extracellular matrix of treated the examples, and fresh tissues were used as controls. Tissue contents of hydroxyproline(alkaline hydrolysis method) and elastin (Fastin Elastin Assay) were assayed respectively in the fresh and decellularized groups (n=10). The vascular wall heat shrinking temperature and mechanical strength were measured to evaluate the tissue stability (n=10). RESULTS: Histochemical and TEM analysis of BJVCs treated with decellularization proved a complete removal of nuclear and other cell components. Tissue collagen was well kept,but elastin was partly lessened. Tissue content of hydroxyproline increased comparatively [(25.73+/-2.97)mg/g vs. (29.25+/-2.99)mg/g, P<0.05] and the elastin content obviously decreased [(159.71+/-21.06)mg/g vs. (134.91+/-35.40)mg/g, P<0.05] in the decellular treatment group compared with the control group. The heat shrinking temperature and tensile stress of decelluarized tissue were lower than those of the fresh tissue[(72.50+/-0.53) degrees C vs. (69.75+/-0.54)degrees C ,P<0.05], [(5.19+/-0.65)MPa vs. (3.13+/-0.94)MPa, P<0.05]. CONCLUSION: The basic framework of extracellular matrix in the decellularized BJVC is partly damaged and tissue stability is reduced. Decellularized BJVC should be further crosslinked before being used as a tissue engineering scaffold for clinical pulmonary artery graft.

[Effect of chemokine interleukin-8, monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 on the angiogenesis of non-small cell lung cancer].

OBJECTIVE: To detect the expressions of chemokines interleukin-8 (IL-8), monocyte chemoattractant protein-1 (MCP-1), and macrophage inflammatory protein-1 (MIP-1) mRNA in non-small cell lung cancer (NSCLC) tissues, to analyze their relationship with microvessel counts (MVC), and their significance in clinic pathologic features of NSCLC. METHODS: In situ hybridization was used to measure the expressions of chemokine IL-8, MCP-1, and MIP-1 mRNA in 40 NSCLC tissues and 10 normal pulmonary tissues, and immunohistochemical staining was carried out to measure the MVC in the above tissues. RESULTS: The positive ratios of IL-8, MCP-1, and MIP-1 mRNA in the 40 NSCLC tissues were apparently higher than those in the 10 normal contrast tissues and the difference was statistically significant. The numbers varied accordingly with the different clinic pathologic features of NSCLC, showing that Group T(3) > Group T(2) or Group T(1), Group III stage> Group II stage> Group I stage Group lymph node and remote transferred > Group non-transferred, and Group of survival time no more than 3 years > Group of survival time more than 3 years. The positive expressions among IL-8, MCP-1,and MIP-1 mRNA and between these and the MVC all had mutually positive correlation. CONCLUSION: Chemokine IL-8, MIP-1, and MIP-1 in NSCLC tissues might cooperate with one another to promote the tumor angiogenesis and affect the progression, metastasis and prognosis of the tumor.

[Relation of tumor associated macrophages and mast cells of tumor interstitial and angiogenesis in non-small cell lung cancer].

OBJECTIVE: To detect tumor microvessel count (MVC), tumor associated macrophage (TAM) and Mast cell (MC) counts, and to explore their correlation and the clinical significance in non-small cell lung cancer (NSCLC). METHODS: Immunohistochemical staining (ABC method) was used to measure MVC and TAM and MC counts in 40 cases of NSCLC tissues and 10 cases of normal pulmonary tissues as contrast. RESULTS: The counts of microvessel, TAMs, and MCs in the NSCLC tissues were apparently higher than those of the normal tissues, and these counts increased with the development of T stage and clinical stage, metastasis of the tumor, and the reduction of the patient survival time. The positive correlation was showed among the counts of microvessel, TAMs, and MCs. CONCLUSION: Tumor interstitial infiltrating inflammatory cells, TAMs and MCs can cooperatively promote the tumor angiogenesis,which associates with the development, invasion, metastasis and prognosis of NSCLC.

[Bovine jugular venous conduit treated with the polyepoxy compound].

OBJECTIVE: To determine the feasibility whether the bovine jugular venous conduit (BJVC) can be fixed with polyepoxy compound (PC). METHODS: Twenty-four BJVCs were divided into 3 groups and fixed with polyepoxy compound (PC group, n = 8), glutaraldehyde (GA group, n = 8), and unfixed group (Control group, n = 8), respectively. The morphologic and mechanical properties of BJVCs in the 3 groups, including thickness, diameter, moisture content, denaturation temperature, tensile strength, elongation at break, and fixation index were measured. The rat subcutaneous model for the assessment of tissue calcification was used. The calcium content in bovine jugular vein patches and valves was determined by flame atomic absorption spectrophotometer. RESULTS: There was no difference in the wall thickness, diameter, and tissue water content between PC and the control group, but significant difference was found between GA and PC groups. The mechanical properties of PC group and GA group were not significantly different, but they were better than those of the control group. GA-fixed BJVC samples showed clear calcification, while PC fixed BJVC were calcified significantly less. CONCLUSION: PC is an effective and suitable choice for the treatment of BJVC since it can effectively preserve the structure and the anti-reflow function of valves in bovine jugular vein and it has better anti-calcification properties.

[Lymphangiogenes and location of tumor lymphatic vessels induced by VEGF-C in primary breast carcer].

OBJECTIVE: To investigate the lymphangiogenesis and location of tumor lymphatic vessels induced by vascular endothelial growth factor C (VEGF-C) in primary breast cancer. METHODS: The expression of VEGF-C mRNA in 89 cases of primary breast cancer was detected by in situ hybridization, and lymphatic vessels with vascular endothelial growth factor receptor-3 (VEGFR-3) were labeled by immunohistochemistry SP method. RESULTS: VEGF-C mRNA expressed in 49 of all 89 cases of primary breast cancer, and the expression rate was 55.06%. The expression of VEGF-C mRNA was positively correlated with lymphatic vessel density (LVD) and axillary lymph node metastasis, LVD and the rate of axillary lymph node metastasis were significantly higher in VEGF-C mRNA positive group than those in the negative group (both P < 0.05). Different levels of lymphangiogenesis took place in all cases of breast cancer, but it was mainly located in tumor stroma, and apparently mature lymphatic vessels were not found in cancer nests. LVD was positive related with the clinical stage and axillary lymph node metastasis in breast cancer; the clinical stage, the LVD, the axillary lymph node metastasis in positive group were higher than those in the negative group (P < 0.05). CONCLUSION: The expression of VEGF-C mRNA is positively correlated with lymphangiogenesis and axillary lymph node metastasis in primary breast cancer. Lymphangiogenesis induced by VEGF-C predominantly takes place in the tumor stroma tissue, and mature lymphatic vessels are not found in cancer nests.

[Morphologic and physiochemical properties of bovine jugular conduit stabilized by dye-mediated poto-oxidation].

OBJECTIVE: To determine the morphologic and physiochemical properties of bovine jugular conduit with valves stabilized by dye-mediated photo-oxidation. METHODS: Twenty-four bovine jugular conduits with valves were divided into 3 groups and treated with dye-mediated photo-oxidation (Group I), glutaraldehyde (Group II) and untreated group (Group II), respectively. Morphologic and physiochemical properties of the 3 groups, including wall thickness, diameter, tissue water content, heat shrinking temperature, breaching strength, and tissue protein extraction assay were studied. RESULTS: There was no difference in wall thickness, diameter, tissue water content, and heat shrinking temperature between Group I and II ,but there was significant difference between Group I and II. The breaching strength of Group I was higher than that of Group IU (P < 0.05), but lower than that of Group II (P < 0. 05). A decrease in extractable tissue protein was found in Group I and II. CONCLUSION: The dye-mediated photooxidation can effectively preserve the structure and the anti-regurgitation function of valves and improve the tissue stability and enhance the tension of bovine jugular conduit with valves.